Because of its high sensitivity, polymerase chain reaction (PCR) is the gold standard for the diagnosis of many infectious diseases, but generally only implemented in well-equipped laboratories. One of the major roadblocks for expanding PCR to point-of-care markets is the lack of simple, robust, single tube PCR designs which preserve its laboratory-based high sensitivity and specificity. In this Fast-Track STTR application BioVentures, Inc. and Vanderbilt University, a nearby research institution, propose to develop a fundamentally different PCR design, hybridization-triggered PCR (HT-PCR). It alters the way PCR cyclic amplification is monitored and controlled, resulting in a design more suitable for underserved point-of-care markets. The HT- PCR technology (co-invented by the applicants) meshes well with BioVentures's successful business model of manufacturing and selling molecular biology reagents and aligns with its strategy to expand into the clinical diagnostic market. Phase I and II Aims evaluate the feasibility and advantages of this approach with studies to detect DNA biomarkers of three major infectious diseases. One of the major impediments to simple, robust, single tube PCR is that an efficient amplification reaction requires a narrow range of thermal and chemical conditions. Point-of-care settings, including walk-in clinics, rural health outposts, and outbreak surveillance by mobile response units, generally lack the stringent sample preparation and controlled environmental requirements available in centralized laboratories. The fundamental limitation with all current PCR designs is that thermal cycling is controlled by pre-determined indirect temperature measurements, yet the PCR product melting step and, more importantly, the primer annealing step, do not always occur at the programmed temperatures. Individual reaction conditions, ambient temperatures, and thermal calibrations create disparities between the expected hybridization state of the product or primers and the actual hybridization state. These disparities are exacerbated in diagnostic settings that are less equipped to precisely control environmental conditions and sample contents, leading to PCR failure, i.e., false negatives. We propose an alternative PCR design that dynamically controls thermal cycling by optically sensing the annealing and melting of mirror-image L-DNA surrogates of the reaction's primers and targets. Because the properties L-DNA enantiomers parallel those of natural D-DNAs, the L-DNA reagents are used to indicate the cycling conditions required for effective primer annealing and product melting during each cycle without interfering with the reaction. A major advantage of this approach is that it enables hybridization- triggered heating and cooling without the need to know reaction temperatures and times. Thus the instrument dynamically adapts to unpredictable thermal and chemical variations. A second major advantage is that the L- DNA surrogates of the PCR product can also be used as controls for reagent rehydration, sample preparation, instrument performance, diagnostic threshold, and correct product formation, enabling well-controlled single- tube analysis of DNA.